Hypoxia is a well-characterized component of the solid tumor microenvironment. Hypoxic tumors are more resistant to radiation therapy and chemotherapy and have a poorer prognosis than better-oxygenated tumors. Classical biochemical studies have shown that exposure of tumor cells to hypoxia results in a pronounced decrease in the rate of protein synthesis, which is reversible upon reoxygenation. This process is hypothesized to constitute an efficient mechanism of cellular energy conservation that is critical for the survival of the tumor cell in this environment of reduced oxygen availability. While significant progress has been made in identifying individual gene products whose synthesis is altered by hypoxia, little is known about the mechanism by which hypoxia induces global downregulation of translation. Phosphorylation of the translation initiation factor eIF2alpha on ser51 plays a key role in the regulation of protein synthesis by other types of stress, such as heat-shock and brain ischemia/reperfusion injury. Preliminary data indicate that exposure of tumor cells to hypoxia increases the levels of eIF2alpha phosphorylation and decreases the rate of protein synthesis. The endoplasmic reticulum-resident kinase, PERK, becomes phosphorylated under hypoxia and may be responsible for hypoxia-induced eIF2alpha phosphorylation. We propose that phosphorylation of eIF2alpha plays a key role in the cellular adaptation to tumor hypoxia. Aim 1 will further characterize the kinetics and establish the oxygen dependency of elF2alpha phosphorylation in transformed and untransformed cells. Aim 2 will investigate whether inhibition of phosphorylation of eIF2alpha is required for inhibition of protein synthesis in cells exposed to hypoxia. In aim 3, genetic and biochemical means will be used to investigate whether the endoplasmic reticulum kinase PERK is responsible for hypoxia-induced phosphorylation of elF2alpha. Aim 4 will examine the consequences of deregulated eIF2alpha phosphorylation and translation on the most important endpoint of cellular adaptation, cell survival/cell death, by determining the short- and long-term viability of tumor cells exposed to moderate or extreme hypoxia. Accomplishing the aims of this application will increase our understanding of the process of cellular adaptation to hypoxic stress. Identification of the key players in this process and examination of the consequences of inhibition of their function on tumor cell survival, may lead to new therapeutic modalities that specifically target this adaptive in hypoxic areas of solid tumors.